Systematic investigation results indicate that the organic Eu3+-complex encapsulated in the functionalized diatomite channels exhibits enhanced luminescence and longer lifetime, owning to the Eu(TTA)3Phen complex interacting with its surrounding silylating agents. The organic Eu3+-complex-anchored porous diatomite hybrid luminescent material was compounded with polyethylene terephthalate (PET) by using a mini-twin screw extruder to prepare a self-supporting film of the hybrid material. Besides, the UV absorption properties of the composite films were investigated. These films will potentially be related to the UV protection of photovoltaic devices.The organic Eu  For example, the cotton fabric modified with ZnO nanoparticles on the surface offers UV-blocking in the wavelength range from 280 nm to 400 nm ,8. Prosatot) [3Phen (ETP), with wide optical absorption [Recently, designing visible luminescence materials by lanthanide complexes has attracted increasing attention for various applications ,16. Eurotot) . In ordesorption . Under Usorption ,19,20. Isorption ,21. Basesorption . Howeversorption ,27. Thersorption . DA is fsorption . Based osorption . Additiosorption ,32,33. Asorption .3+-complex. More importantly, this organic-inorganic hybrid luminescent material was applied as UV absorbance additive for PET composite film, and exhibited excellent UV-protection properties.In this work, the ETP complex was anchored in the pores of DA via the grafted (3-aminopropyl) trimethoxysilane (APTMS) through impregnation method, which improves the photophysical properties of the complex. Compared with the pure ETP complex, the luminescence properties of the sample encapsulated in APTMS-modified DA are greatly improved. Moreover, the existence of APTMS is much helpful to improve the luminescence properties of the organic Eu2.2.1.3•H2O, 25%), toluene (99.5%) and ethanol (95%) were purchased from Sinopharm Chemical Reagent Co., Ltd. Europium chloride hexahydrate , 1,10-phenanthroline  and 2-thenoyltrifluoroacetone  were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Polyethylene terephthalate (PET) was purchased from Sinopec Yizheng Chemical Fiber Co., Ltd.Diatomite (DA) was obtained from Lumino food grade diatomaceous earth . (3-Aminopropyl) trimethoxysilane  was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. Ammonium hydroxide  was added to the above solution and the mixture was stirred. Then, the mixture was refluxed for 10 hours under a static dry nitrogen atmosphere. After the silylation reaction, the product (APTMS-DA) was collected by centrifugation, washed thoroughly with toluene and ethanol and then dried at 602.4.3 solution was added into the above mixture. After the pH was adjusted to 6 ~ 7 with ammonium hydroxide, the solution was stirred for 5 h at room temperature. The APTMS-DA hybrid luminescent material was collected and washed several times with ethanol to remove the dye until no luminescence was observed in the washing solution. The final deposit was dried at 60°C. The preparation of ETP-DA was similar to the method described above.First, 10 mg of APTMS-DA sample was added to ethanol and stirred. Then, the same volume of TTA/ethanol and Phen/ethanol solution with the concentration of 0.03 mol/L and 0.01 mol/L, respectively was dropped into the above solution and stirred for 10 min. Finally, the EuCl2.5.°C for 24 h to remove moisture. Then, the blending of ETP-APTMS-DA and PET were melt using a mini-twin screw extruder (HAAKE Rheomex PTW16). The content of ETP-APTMS-DA was 0.1%, 0.2% and 0.3% by weight. PET/ETP-APTMS-DA composite films were prepared using a hot press.The products obtained in the previous step and PET were vacuum dried at 802.629Si NMR, Bruker Avance III HD 400 MHz). Optical absorption spectra were recorded with an ultraviolet and visible spectrophotometer . The luminescence properties of the samples were studied with a fluorescence spectrometer (FLS 980-STM). The information on chemical bonds of the samples were investigated by X-ray photoelectron spectroscopy . Elemental analysis was carried out via inductively coupled plasma emission mass spectrometry . The specific surface area and pore size distribution of the samples were measured with an automatic specific surface area and pore analyzer (Tristar3000). A possible UV-protection mechanism of PET composite film is shown in The morphologies of the samples were observed via field emission scanning electron microscopy . The composition and crystal structure of the samples were characterized by X-ray diffraction . The functional groups of the samples were explored via Fourier transform infrared spectrometry . The structure and the attachment modes to the diatomite surface of the siloxane molecules were investigated by nuclear magnetic resonance  peaks at the range of 3100–3750 cm−1 and 1639 cm−1 present the stretching vibration and bending vibration, respectively, which derived from the physical adsorption water and the free hydroxyl groups on the surface area of the DA [−1 can be attributed to the stretching and bending vibrations of primary amine. Moreover, the bands at 2939 (asymmetric stretching), 2839 (symmetric stretching), 1463 and 1411 cm−1 correspond to CH2 groups. The FTIR spectrum of APTMS-DA is similar to that of the DA. Whereas the appearance of C-H vibration at 2935, 2881, 1492 and 1438 cm−1and the N-H bending mode at 1560 cm−1 both prove the existence of organic moieties on the surface of the diatomite after modification. Therefore, it can be concluded that the surface of DA has been successfully modified by APTMS.As shown in f the DA . Actuall−1 after the addition of Eu3+-complex to APTMS-DA. The absorption peaks of TTA at 1659 cm−1, 1638 cm−1 and 1359 cm−1 correspond to the vibration absorption peaks of C = O. The absorption peaks of Phen at 1616 cm−1 represents the vibration absorption peaks of C = N. Compared with TTA, the absorption intensity of C = O bond in ETP decreases and the absorption peak of C = O shifts to 1627 cm−1. Meanwhile, a new peak appears at 580 cm−1, which can be assigned to the stretching vibration of Eu-O [3+. Besides, the characteristic absorption peak corresponding to the C-N bond shifts from 1616 cm−1 to 1601 cm−1. Therefore, it can be speculated that a chemical bond is formed between Phen and Eu, and then the ETP is successfully synthesized. The spectrum of ETP-APTMS-DA shows both characteristic bands of the ETP complex and APTMS-DA. Based on the above characterization data, ETP complex is successfully attached on the surface of APTMS-DA.Through the FTIR analysis of the hybrid materials of ETP and ETP-APTMS-DA composites, the chemical bonds and functional groups were characterized in detail to prove the presence of ETP complexes in the APTMS-DA, which benefits to the FTIR results shown in  of Eu-O . These b29Si NMR test was carried out and the results of the APTMS-modified DA (APTMS-DA) further confirm the successful reaction of APTMS with the surface of DA, compared to bare diatomite (DA) and APTMS used as the controls. The results are presented in 0 structure in which no ethoxy group has been substituted. The three resonance signals at −90.14 ppm, −98.92 ppm and −109.53 ppm detected in the DA are corresponding to Q2, Q3 and Q4, respectively [3 structure) appears but the characteristic peak of APTMS disappears confirming that APTMS is not physically adsorbed on the DA surface and no excess siloxane molecules. The bonding modes of the APTMS to the DA surface are shown in Furthermore, ectively . This isectively . For theElemental analysis was used to determine the grafting ratio of APTMS grafted on the surface of DA and the related data are listed in N is the percentage of N element of APTMS grafted on the surface of DA that can be obtained by subtracting the content of elemental N in DA from APTMS-DA. As can be seen from N of 1.78%. The graft ratio of APTMS was calculated to be 1.27 mmol/g.In the above formula, M corresponds to the effective molar density of APTMS grafted onto the DA surface. W3.3.3+-complex anchoring into the channels of APTMS-modified as well as unmodified DA, so the polarity of the surrounding environment exhibits changes, which are similar to previous reports [ reports ,20,23,243.4.3+-complex in ETP-DA and ETP-APTMS-DA were broadened and shifted toward shorter wavelength, which can be attributed to the changes in the ligand environment. This is also proved by the UV-vis absorption spectra. To compare the performance of different luminescent samples, the emission spectra were obtained using their maximum excitation absorption band under the same condition. The result of PL spectra (5D0-7F2 emission which results in ‘europium red’ luminescence. The transition of Eu(III) ion  is well known to be sensitive to the coordination environment of the Eu(III) ion. When the interaction of the ETP complex with its local chemical environment is stronger, the ETP complex becomes more asymmetric and the intensity of transition becomes higher. The 5D0-7F2/5D0-7F1 emission intensity ratios of ETP, ETP-DA and ETP-APTMS-DA are 8.2, 8.3 and 8.6, respectively, which reflects the increase in the intensity of the 5D0-7F2 emission band and the decrease in other emission bands due to the increase in electric dipole interaction [5D0-7F2 transition of the ETP and ETP-APTMS-DA samples is 8.30 and 9.48, respectively. Therefore, the bandwidth of the emission spectrum of the complex with the modified DA is significantly different with that of the pure complex. Besides, PL intensities of the three samples are increased with the order, i.e., pure ETP < ETP-DA < ETP-APTMS-DA. On the other hand, the emission quantum yields (Φtot) are determined by the following procedure: when the emission wavelength is at 613 nm for all samples, the excitation wavelengths are verified at 370 nm, 346 nm and 346 nm for ETP, ETP-DA and ETP-APTMS-DA, respectively. The scanning wavelength range (200–800 nm) is used to get the absolute quantum yield. The emission quantum yields (Φtot) are 5.19%, 6.41% and 17.52% corresponding to ETP, ETP-DA and ETP-APTMS-DA. With the similar sequence, the quantum yield of the ETP-APTMS-DA is higher than that of others. The changes can be rationalized on the basis of the interaction between NH2 groups and the ETP. Similar phenomenon and conclusion also are observed in other study [Luminescence properties of pure ETP, ETP-DA and ETP-APTMS-DA samples are shown in  spectra ) shows teraction . Meanwhier study .3+ ions related to 5D0-7F2 transitions in ETP, ETP-DA and ETP-APTMS-DA samples are shown in The luminescence decay curves of Eu3+ ions with TTA and Phen (i.e. ETP). The different decay behaviours in different samples are due to the existing vicinity difference of Eu3+ complex in ETP, ETP-DA and ETP-APTMS-DA. It can be seen that the lifetime of ETP-APTMS-DA is longer than ETP and ETP-DA. Indeed, the interaction between the complex and the functional group grafted on the DA surface changes the intensity and relaxation time of the molecular vibration. The short lifetime of ETP-DA can be speculated to relate to the quenching influence of O-H groups on the surface of DA on the complex [Luminescence decay components and their lifetime values are listed in  complex ,20,22–242 group on the DA surface [2 network structure), while the ETP-APTMS-DA has another lower energy of 102.47 eV corresponding to SiOx-C. This result also proves that APTMS successfully modified DA.As shown in  surface , which e3+ ions in different samples were obtained by ICPMS measurement, to be 0.58% for ETP-DA and 1.48% for ETP-APTMS-DA, which reflects the strength of the interaction between ETP complex with APTMS-DA is stronger than that between ETP complex with DA.The contents of the Eu3+-complex has been incorporated to porous channels of DA. The N2 adsorption-desorption isotherm and its BJH pore size distribution for DA, APTMS-DA, ETP-DA, and ETP-APTMS-DA are shown in The evaluations of channel size were measured to confirm that the anchored organic Eu3.5.PET composite film can not only absorb ultraviolet rays for protection but also utilize the UV light by converting UV light to emission energy see  inset. T4.3+-complex-anchored porous diatomite channels that enable UV protection and downconversion in hybrid PET-matrix film. We found that ETP anchored at APTMS-modified DA surface, and exhibited the long lifetime and the high luminescence intensity. The XPS and UV-vis absorption spectra demonstrated that the emission properties of the organic Eu3+-complex were greatly influenced by its vicinity interactions in ETP complex and APTMS-DA. Generally, the photoluminescence properties of ETP complexes anchored in APTMS-DA were improved considerably in comparison to other two samples. On the other hand, the transparency of pure PET film is approximately 90% at 500 nm without significant UV absorption for UV-A, whereas the UV-A absorption of ETP-APTMS-DA doped PET film is ~40%. Therefore, ETP-APTMS-DA can be used as an excellent UV blocker in PET transparent films. As expected, this UV-protection flexible film will potentially apply for the large area flexible polymer solar cell to enhance efficiency and to extend service life.In this work, we put effort to explore the new usage of porous natural material, diatomite, for high efficient luminescent materials and further for UV protection in soft host. The most important innovation is the successful synthesis of organic Eu


Objectives: This study aimed to detect the time window of vascular normalization during anti-vascular treatment using intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI). Simultaneously, we evaluated the tumor invasiveness and vasculogenic mimicry and performed synthetic assessment of treatment efficacy of angiogenesis inhibitor combined with conventional chemotherapy using IVIM-DWI.Materials and Methods: HCT116 cells were subcutaneously administered into the right flank of BALB/C nude mice to build a colon cancer xenograft model. Thirty-two tumor-bearing mice were randomly divided into four groups and intraperitoneally administered with normal saline (Group A or control group), bevacizumab (Group B), oxaliplatin monotherapy (Group C), and oxaliplatin combined with bevacizumab (Group D). The IVIM-DWI was performed on days 0, 3, 6, 9, 12, and 15 after the treatments. Another 51 tumor-bearing mice were included in the pathological examinations. α-Smooth muscle actin (SMA) and CD31 double-staining, periodic acid-Schiff (PAS) and CD31 double-staining, hematoxylin and eosin (HE), Ki-67, and E-cadherin staining were performed. The tumor growth and dynamic change of each parameter were noted.Results: The mice in Group D manifested the smallest tumor volume and highest tumor inhibition rate. Microvessel density was significantly decreased but accompanied by increased vasculogenic mimicry after antiangiogenic treatment. The trend was reversed by oxaliplatin treatment. Treated with bevacizumab, the vessel maturity index shared a similar trend with D* and f-values during days 3–12, which slowly increased from days 0 to 9 and then decreased briefly. D-value significantly correlated with vasculogenic mimicry and Ki-67, while D* and f-values showed positive correlations with microvessel density and E-cadherin, an indicator of epithelial–mesenchymal transition.Conclusion: Oxaliplatin performed an inhibited effect on vasculogenic mimicry. Bevacizumab can enhance the tumor chemotherapy through vascular normalization within a transient time period, which can be detected by IVIM-DWI. D* and f-values are able to predict the tumor invasiveness while D is superior in reflecting vasculogenic mimicry and Ki-67 expression during antitumor treatment. Tumor vascularization plays an important role in tumor growth, invasion, and metastasis. The lack of adequate oxygen supply for tumor growth will induce the formation of a hypoxia microenvironment. In order to counteract the severe condition, the tumor cells and tumor microenvironment will upregulate the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α (HIF-1α) and subsequently perform a series of adaptive changes. Initially, a great amount of newborn vessels are formed, with high leakage due to the absence of smooth muscle cells and pericyte coverages, and incomplete basement membrane. The immature vessels deliver inadequate nutrition and oxygen but elevate the interstitial fluid pressure, which will aggravate the tumor hypoxia and lead to tumor cell transformation into an aggressive phenotype. Epithelial–mesenchymal transition (EMT) will be induced by activating HIF-1α , 3.Antiangiogenic therapy has been widely used in the clinical treatment of various solid tumors. It is a promising therapy that targets the VEGF which plays an important role on the neovasculature formation. Jain  proposedOxaliplatin is the third-generation platinum anticancer drug. It has an active antitumor effect against a variety of tumors and performs a synergistic effect with 5-fluorouracil . HoweverMonitoring the tumor microenvironment is an important direction of current antitumor therapy, including the degree of tumor hypoxia, EMT, and various compensatory angiogenesis pathways, which have close relationships with each other and determine the efficacy of chemoradiotherapy or immunotherapy. Currently, how to effectively monitor the dynamic change of EMT and VM as well as vascular normalization and therapeutic efficacy is the key point of this research. Although dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is the gold standard for evaluating vascular perfusion and permeability in imaging, continuous enhanced imaging in a short time requires multiple injections of contrast agent, which poses a certain risk to the organisms. Intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI) is a noninvasive technique which was first proposed by Le Bihan et al.  in 1986.2 atmosphere.We obtained the human colon cancer HCT116 cell line from the Pharmaceutical College of Jinan University. They were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in 5% CO6/ml) mixed with 10 ml of Matrigel were subcutaneously injected into the right flank of the mice for creating colon cancer-bearing mice. At last, 83 tumor-bearing mice were selected for imaging (n = 32) and pathological examinations (n = 51). The mice for the MRI examinations were randomly divided into four groups and then intraperitoneally injected with normal saline (Group A or control group), bevacizumab , oxaliplatin , and oxaliplatin combined with bevacizumab (Group D) on days 1, 4, 7, 10, and 13. The tumor volume was calculated using the following formula: (a2 × b × 0.5) mm3, where a refers to the smaller diameter and b is the diameter perpendicular to a, measured with a slide caliper  were purchased from Beijing Vital River Laboratory Animal Technology Corporation  and kept in specific pathogen-free conditions. HCT116 cells  of 2. T2WI was performed with fast recovery FSE and TR/TE of 2,140/82.3 ms. The other parameters were in accord with T1WI. The IVIM-DW MR images were obtained using a single-shot, echo-planar imaging pulse sequence with chemical shift-selective saturation technique for fat suppression. Three orthogonal directions were set as the diffusion gradients with 13 b-values: 0, 25, 50, 75, 100, 150, 200, 400, 600, 800, 1,000, 1,200, and 1,500 s/mm2, TR/TE of 4,200/101.7 ms, NEX of 3 for each b-value, matrix size of 96 × 128, and FOV of 7.0 × 5.6 cm2. This sequence took 7 min 20 s, with six slices covering the whole tumor.The MRI examination was conducted on a 1.5 T Signa HDxt superconductor MR system  with an eight-channel wrist coil. The four groups of mice were intraperitoneally administered with 0.1% pentobarbital solution and scanned in the supine position prior to and on days 3, 6, 9, 12, and 15 after therapy (0 = (1 – f) × exp(–bD) + f × exp(–bD*). SI0 refers to the mean signal intensity of the region of interest (ROI) for a b-value of 0 s/mm2, while SI refers to the signal intensity for higher b-values. B-value means the diffusion sensitivity coefficient. D-values represent the pure diffusion of water molecule, also known as true diffusion coefficient. D* values refer to pseudo-diffusion, which calculate the amount of microcirculation perfusion. F-value is the perfusion fraction, standing for the percentage of microcirculation perfusion among the signal decay from the total diffusion effect. A segmented method was used to fit the bi-exponential IVIM model. First, b < 200 mm2/s was referred to as low b-value, mainly reflecting the pseudo-diffusion. The data in this range were fitted to the bi-exponential model for acquiring D* and f-values. Then the data of b-values higher than 200 mm2/s were used to obtain D-value using a mono-exponential model because the pseudo-diffusions from blood flows were negligible in this region (0 = exp(–bD) since the f and D* values verge to zero at the b-values higher than 200 mm2/s. Structural T2WI was referenced to plot three ROIs at the largest cross section and adjacent slices of the tumor. The ROI was also indicated in the MRI figures with a white circle. We averaged the three ROIs as the final values.All IVIM-DWI data were transferred to a dedicated post-processing workstation  using the Functool-MADC software. The equation of the bi-exponential model was expressed as SI/SIOut of the 51 mice that were included for pathological analysis, three of them were served for baseline histological analysis (day 0), and for the other 48 mice, each three were sacrificed and sampled on days 3, 6, 9, and 12 for each subgroup of histological analysis. Besides, the mice for the MRI examinations were all sacrificed on day 15. Their tumor samples were regarded as the pathological results. Hematoxylin and eosin (HE) staining, Ki-67 and E-cadherin immunofluorescent staining, periodic acid-Schiff (PAS) and CD31 immunohistochemical double-staining, and α-smooth muscle actin (SMA) and CD31 immunofluorescent double-staining were performed to analyze the pathological changes. The antibodies were all purchased from Servicebio Technology Co., Ltd. . Ki-67 is a proliferation marker reflecting the degree of proliferation activity of tumor cells. VM can be recognized by positive PAS staining and negative CD31 staining, a marker of endothelial cells . The tumpost hoc test. The correlation strengths between imaging parameters and pathological results on day 15 were calculated using Pearson correlation analysis. P < 0.05 was regarded a statistical difference. A Pearson coefficient larger than 0.8 was considered highly correlated, while the coefficient between 0.5 and 0.8 was considered moderately correlated. The line chart and scatter chart were plotted with GraphPad Prism 5.01 .We used SPSS 13.0 software  to calculate statistical results. The data distribution type was confirmed using Kolmogorov–Smirnov test. The numeric result with normal distribution was shown as the mean value with standard deviation (SD). The difference within and between groups were compared by one-way analysis of variance (ANOVA) with Student–Newman–Keuls q test as a No mice died or showed obvious abnormal reactions during the treatment. An obvious inhibitory effect on tumor growth was shown as early as 6 days after the treatment with a much slower growth rate in tumor volume during treatment. The mice treated with the combination therapy manifested the smallest tumor volume and highest tumor inhibition rate at the final time point. The mice treated with oxaliplatin showed smaller tumor volume than that with bevacizumab and compared with the control group. The tumor inhibition rates on day 15 were 35.5%, 49.6%, and 68.6% in Groups B, C, and D, respectively.D, D*, and f-values in Group D is manifested in One group of representative conventional T1WI, T2WI, and pseudo-color maps of On conventional images, the tumor showed slow growth with little tumor volume change during days 3–15 in Group D. Small patchy high signals indicating liquefactive necrosis were observed in the tumor core on T2WI as early as day 6 after the treatment in this group. For Groups B and C, the tumor volume moderately increased, and multiple scattered high signals were revealed on T2WI on days 12 and 15. For Group A, the tumor area showed a rapid increase with an obvious high signal on T2WI on day 12.D-value showed a slow decline , while D*  and f-values  gradually increased until day 15 in Group A, which may be caused by the physiological growth of the tumor with crowded cellularity and increased demand of blood supply over a long period of time.For the MR indicators, D-value slowly decreased over time and started to rise on day 6 , indicating that the restricted diffusion of water molecules was relieved due to the treatment. D*  and f-values  gradually increased during day 0 to 9, representing an augment of tumor perfusion due to vascular normalization. They decreased between day 9 and 12, followed by a slight upward trend after day 12. The delayed antiangiogenic effect and the recession of vascular normalization may account for the subsequent changes on tumor perfusion.For bevacizumab monotherapy, D-value maintained a slow upward trend and was higher than those in Groups A and B , suggesting an improved treatment efficacy. D*  and f-values  initially increased and then started to decrease on day 6. These may be a subsequent change due to tumor necrosis.For oxaliplatin monotherapy, D-value increased significantly after 6 days and reached a peak on day 15, which was also higher than that of the other groups. D*  and f-values  increased to a peak on day 9 but lower than those of Group B, and then fell back until day 15. The trend indicated a similar anti-vascular effect as in Group B.For combination therapy, The mean values of pathological indicators in Groups A, B, C, and D at each time point are shown in The tumor cells in Group A were dense. A few necrosis areas were observed as the tumor grew. In Group B, a small amount of necrosis and cell apoptosis appeared in the tumor core. More karyopyknosis and nuclear fragmentation with homogeneous red-stained areas were observed in the later time points. In group C, small patches of hemorrhage and necrosis appeared on day 3, which mainly distributed in the tumor core. More necrosis areas were observed in Group D, especially on days 12 and 15.Regarding the double-staining section of α-SMA and CD31 , elevateRegarding the double-staining section of PAS and CD31 , both GrGroup D showed the lowest Ki-67 expression on day 15 compared to the other groups, indicating that the combination therapy could have the most remarkable suppressing effect on tumor cell proliferation activity .The E-cadherin expression rapidly increased in both Groups B and D, especially in Group D, indicating more stable intercellular connectivity and lower likelihood of EMT after treatment. A mild increase was also observed in Group C, but a slow decline was observed in Group A with no statistical difference .f-value had the highest correlation with MVD , followed by D* and D-values. D-value showed the strongest correlation with VM , followed by f and D* value. In reflecting cell proliferation activity, D-value was most strongly correlated with Ki-67 , followed by D* and f-value. In assessing metastatic potential of tumor cells, D* value was most highly correlated with E-cadherin staining , followed by f and D-value. In predicting tumor growth, D-value had the highest negative correlation with tumor volume , followed by D* and f-value.The correlation coefficients between imaging and pathological indicators are listed in D-value significantly correlated with vasculogenic mimicry and Ki-67, while D* and f-values showed positive correlations with microvessel density and E-cadherin. The vascular normalization and concomitant microenvironment changes can be monitored in vivo by the noninvasive IVIM-DWI technique.In this study, we continuously monitored the tumor cellularity and microcirculation perfusion changes on a colon cancer model during antiangiogenesis combined with oxaliplatin treatment by IVIM-DWI. We found the combination therapy had the most significant inhibiting effect on tumor growth. The formation of vasculogenic mimicry was stimulated by single bevacizumab treatment, but was suppressed to a certain extent by oxaliplatin treatment. The tumor growth was most inhibited in Group D with the tumor inhibition rate of 68.6% on day 15, followed by oxaliplatin and bevacizumab monotherapy. Besides, no obvious abnormal reaction or death occurred in the mice in the combination setting. Both pathological staining and conventional MRI suggested a large area of necrosis and decreased Ki-67 expression across the tumors at a molecular level. The higher Ki-67 expression had been reported to be correlated with a higher risk of metastasis and recurrence, poorer clinical and pathological response, and worse survival . It has Hypoxia has emerged as a crucial factor in tumor pathophysiology and closely correlated with tumor invasiveness and formation of new angiogenic modes. In this study, we found one important phenomenon that VM significantly increased after bevacizumab treatment though MVD was reduced due to the antiangiogenic effect, which may account for the failure of bevacizumab monotherapy. However, the number of VM was significantly reduced in the single or combined oxaliplatin scheme, suggesting an inhibited effect on VM by a chemotherapeutic drug. This may evidence the synergistic effect of bevacizumab in the combined setting. Essentially, the VM passage is reconstructed from the crowded tumor cells due to insufficient blood supply, which are sensitive to oxaliplatin. This may imply the great value of research on therapeutic drugs that target VM formation in the future.D-value reflects the diffusion dynamics of water molecule, which may be limited in the crowded tumor cellularity with a high proliferative activity. Increased Ki-67 expression suggested higher cellularity and active vascular hyperplasia  and Ki-67 (r = −0.781), indicating D-value can be used to predict treatment efficacy. Furthermore, both D* and f-values shared a similar trend with VMI during day 3 to day 12 after anti-vascular treatment and had high correlations with VMI (r = 0.653 and 0.606) and MVD (r = 0.784 and 0.614), indicating that D* and f values can be used to monitor the process of vascular normalization based on perfusion changes. Interestingly, D-value rather than D* or f-value showed the highest correlation with VM. Though VM is a kind of vessel-like structure, its formation is closely correlated with tumor hypoxia and crowded cellularity and thus can be reflected by D-value. On the other hand, D* and f-values had higher correlations with E-cadherin. Previous study had reported that tumor hypoxia under lower perfusion induces EMT in several types of cancer including prostate cancer, oral cancer, and breast cancer (The tumor volume of each treatment group was much smaller than that of the control group after treatment, suggesting a considerable tumor-suppressive effect. However, evaluation on tumor volume often lags behind functional changes such as tumor perfusion and diffusion after treatment. In this study, we used IVIM-DWI to dynamically monitor the microenvironmental changes and found that both diffusion and pseudo-diffusion parameters demonstrated significant changes than tumor volumes as early as day 3 after treatment. erplasia . In thist cancer . An incrThere are some limitations in this study. First, hypoxia is an important characteristic of tumor microenvironment. However, we have not performed a hypoxia imaging which was introduced by Shi et al. . Second,D* and f-values are able to predict the tumor invasiveness, while D is superior in reflecting vasculogenic mimicry and Ki-67 expression.In conclusion, IVIM-DWI can well detect the microenvironment changes based on diffusion and perfusion characteristics and reflect the efficacy of cancer treatment. From the study, bevacizumab is confirmed to induce vascular normalization and have a significant inhibitory effect on MVD but could stimulate the formation of VM that has a close relationship with tumor invasiveness. The stimulation of VM can be reversed by the treatment of oxaliplatin to a certain extent. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institutional Animal Ethics Committee of Jinan University. Written informed consent was obtained from the owners for the participation of their animals in this study.HH, SZ, and CX contributed to conceptualization. ZL and JL contributed to the animal experiments. CP and WD contributed to MRI examinations. JL and ZL contributed to pathological analysis. JL contributed to writing the original draft preparation. CX contributed to writing, reviewing, and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Frailty is a multidimensional syndrome that leads to an increase in vulnerability. Previous studies have suggested that frailty is associated with poor health-related outcomes. For frailty screening, the Clinical Frailty Scale (CFS) is a simple tool that is widely used in various translated versions. We aimed to translate the CSF into Korean and evaluated its contents and concurrent validity.Translations and back-translations of the CFS were conducted independently. A multidisciplinary team decided the final CFS-K. Between August 2019 and April 2020, a total of 100 outpatient and inpatient participants aged ≥65 years were enrolled prospectively. The clinical characteristics were evaluated using the CFS-K. The CFS-K scores were compared with those of other frailty screening tools using Pearson’s correlation coefficient and Spearman’s rank correlation. The area under curve (AUC) for identifying the Eastern Cooperative Oncology Group Performance Status (ECOG PS) grade 3 or more was calculated for the CFS-K and other screening tools.p = 0.013) and low score on the Korean version of the Mini-Mental State Examination (p < 0.001) were significantly associated with high CFS-K scores, except for those assigned to scale 9 . The CFS-K showed a significant correlation with other frailty screening tools , except in the case of those assigned to scale 9 . In comparison with other scales, the CFS-K identified ECOG PS grade 3 or more with the best performance (AUC = 0.99). Patients assigned to scale 9 on the CFS-K  had similar frailty scores to those assigned to scale 4 (vulnerable) or 5 (mildly frail).The mean age of the participants was 76.5 years , and 63 (63%) participants were male. The mean CFS-K was 4.8 . Low body mass index (In conclusion, the CFS-K is a valid scale for measuring frailty in older Korean patients. The CFS-K scores were significantly correlated with the scores of other scales. To evaluate the predictive and prognostic value of this scale, further larger-scale studies in various clinical settings are warranted. Frailty is a multidimensional syndrome involving loss of reserves  accompanied by an increase in vulnerability to increased dependency and/or mortality when exposed to a stressor , 2. FraiA recent consensus conference which was attended by the international societies and experts in the area of frailty recommended screening for frailty in all older persons and individuals with significant weight loss due to chronic disease . They alIn Korea, The Eastern Cooperative Oncology Group Performance Status (ECOG PS) scale, the Korean version of FRAIL (K-FRAIL), Korean Cancer Study Group Geriatric Score (KG-7), and Korean Frailty Index are commonly used to assess frailty due to lack of appropriate measures –25. HoweWe prospectively enrolled 100 patients aged ≥65 years who visited an outpatient clinic or were admitted to the general ward or intensive care units of the Samsung Medical Center and Samsung Changwon Hospital between August 2019 and April 2020. The patients were eligible to participate if they or their guardians, who were closely involved in their care, gave informed consent to measure frailty. Patients diagnosed with dementia were excluded. The Institutional Review Board of the Samsung Medical Center (IRB No. 2019–02–028-004) and Samsung Changwon Hospital (IRB No. 2019–06-003) approved this study, and each participant provided informed written consent.Original CFS in English consists of a scale from 1 (very fit) to 9 , which is scored by clinical judgment; hence, the last group is technically not frail  To assess the baseline cognitive function, we used the Korean version of the Mini-Mental State Examination (K-MMSE) . Other dTo examine the concurrent validity, we used the ECOG PS, K-FRAIL scale, KG-7, and Korean Frailty Index –25, 28. p-values to compare the AUC of CFS-K with AUC of other frailty screening tests. The significance level was set at 0.05. All analyses were performed using STATA version 15 .Data analyses included descriptive statistics  and statistical analyses for assessing frailty. In order to measure the CFS-K’s concurrent validity, Pearson’s coefficients for correlation between CFS and the other scales including the KG-7, K-FRAIL, and Korean Frailty Index, and Spearman’s rank-order for correlation between CFS and ECOG PS were computed. In addition, we calculated the sensitivity and specificity of identifying ECOG 3 or more using the area under the curve (AUC). The performance for identifying frailty was compared between CFS-K and the K-FRAIL, KG-7, and Korean Frailty Index, with Bonferroni’s correction to adjust for multiple comparisons. For the analyses, KG-7 was recorded in reverse to achieve the same direction scores. We used the two way sided n = 10, 10.0%) and inpatient  participants were recruited at two medical centers. The mean age of the study participants was 75.6 years old and 63.0% were men. The mean body mass index was 21.7 kg/m2 . Among the participants, the common comorbidity was chronic lung disease including chronic obstructive pulmonary disease, asthma, and interstitial lung disease (44%), followed by hypertension (38%) and cancer (32%). For the 90 inpatients, the primary reason for admission was pneumonia (42.2%) followed by cancer-related management (24.4%). Of the 10 outpatients, 9 visited for pulmonary disease (90%), and one visited for cardiovascular disease (10%). K-MMSE was measured in 96 (96%) patients and the mean score of the K-MMSE was 22.2 (SD 7.2).The participants’ characteristics are demonstrated in Table P for trends < 0.001). The body mass index of patients assigned to scale 9  was higher than that of patients assigned to scale 2 (well) and the K-MMSE score (21.0 (8.7)) of patients assigned to scale 6 (moderately frail) (18.9 (5.9)) was higher than that of patients assigned to other scales.All of the participants completed the CFS-K, and the mean score was 4.8 SD, 2.5). The characteristics of the participants are grouped by CFS-K scale (Table .5. The cThe frailty scores by CFS-K are summarized in Table p = 0.08), K-FRAIL , and Korean Frailty Index   curve for identifying ECOG PS grade 3 or more, the CFS-K showed better performance (AUC = 0.99) than the KG-7  Fig. . In addiIn this study, we translated the CFS into Korean and evaluated the content and concurrent validity by comparing it with other scales, namely the ECOG PS, K-FRAIL, KG-7, and Korean Frailty Index. The patients were subjected all of the scales regardless of age or sex. High scores on the CFS-K were correlated to low body mass index and low K-MMSE score. The CFS-K scores were significantly correlated with the scores of other scales and showed the best assessment of frailty.The CFS-K was found to be a useful screening tool of frailty in Korean older patients. In this study, the newly translated Korean version of CFS recognized frail patients more effectively than other scales. Previous studies have suggested the CFS is a useful screening tool based on clinical judgement for measuring frailty , 14. theInterestingly, the body mass index and K-MMSE score showed significant differences across the CSF-K scales. The relationship between frailty and sarcopenia has been reported in several studies , 30; thiThe patients assigned to scale 9  showed unique characteristics. Because of the definition, for patients who were not evidently frail but had less than 6 months’ life expectancy, the scores of frailty indexes were between scale 4 (vulnerable) and scale 5 (mildly frail) and the ECOG PS score also ranged from 0 to 3; their body mass index and K-MMSE scores were also relatively high. In this study, the patients diagnosed with advanced solid or hematologic malignancy with high tumor burden were assigned to scale 9 . Nowadays, the life expectancy is increasing due to improvement in cancer treatment, organ transplantation, and critical care with organ-supporting systems –39. FurtThis study has some limitations. First, the validation of the CFS-K was performed with a relatively small number of participants. Second, this study included outpatients and inpatients, not a community-based population. Moreover, only patients who gave consent were enrolled. These factors might have caused selection bias. Further large-scale studies with general population and patients in various clinical settings are warranted.In conclusion, the CFS-K is a valid scale for measuring frailty in older Korean patients. The CFS-K scores were significantly correlated with the scores of other scales. To evaluate the predictive and prognostic value of this scale, further larger-scale studies in various clinical settings are warranted.


Background: In the context of declining smoking rates in Estonia, this study aims to analyze the recent trends in e-cigarette use and its associations with smoking status and sociodemographic factors. Methods: Nationally representative data from biennial cross-sectional health surveys in 2012–2018 (n = 9988) were used to describe the prevalence of smoking and e-cigarette use by smoking status in Estonia. Multivariate logistic regression analysis was used to describe the sociodemographic patterns of e-cigarette use in three subgroups: the general population, smokers, and ex-smokers. Results: The prevalence of current smoking decreased from 45.4% in 2012 to 31.5% in 2018 among men and from 26.6% to 20.0% among women. At the same time, e-cigarette use in the general population had increased to 3.7% among men and to 1.2% among women. The increase in the prevalence of e-cigarette use was statistically significant among men in the general population, smokers, and ex-smokers, but non-significant among women. In addition to period effects, e-cigarette use was patterned by age, gender, and education. Conclusion: In 2002–2018, the e-cigarette use had increased but smoking had decreased in Estonia. A timely and targeted tobacco policy may alleviate the harm of e-cigarette use from the public health perspective. Estonian tobacco legislation has witnessed several significant changes over the past decades that have had a positive effect on overall smoking rates. Since the implementation of the Tobacco Act in 2001 and its later revisions  suggestsElectronic nicotine delivery systems (ENDS), commonly known as e-cigarettes, have become increasingly popular in recent years . As theyWhile there is lack of consensus on the long-term harms or benefits of e-cigarettes in terms of smoking uptake or cessation, the majority of e-cigarette users are either current or ex-smokers ,7,8. AvaThe present study is motivated by a hypothesis that the previously described decline in smoking prevalence could—at least partly—be explained by the increasing prevalence of e-cigarettes. Therefore, we will describe the recent trends in e-cigarette use among the adult population of Estonia and analyze its associations with smoking status and sociodemographic factors.Data from nationally representative cross-sectional postal surveys of Health Behavior among Estonian Adult Population in 2012–2018 were used for this analysis. All surveys were approved by the Tallinn Medical Research Ethics Committee. The detailed description of survey methodology is available elsewhere . This stDuring 2012–2018, the prevalence of current smoking among 16–64-year-old men declined from 45.4% (95% CI 42.8–48.0) to 31.5% (95% CI 28.9–34.1). Among women, the change was slightly smaller, with 20.0% (95% CI 17.9–22.3) being smokers in 2018 compared to 26.6% (95% CI 24.4–28.9) in 2012. The decline in smoking rates was statistically significant for both genders.The same period saw a statistically significant increase in e-cigarette use . Among mAmong the smoking population, a statistically significant increase in e-cigarette use during 2012–2018 was found only for men. In 2018, e-cigarettes were regularly used by 6.3% (95% CI 4.2–9.0) of smoking men compared to 2.3% (95% CI 1.3–3.7) in 2012. Among smoking women, e-cigarette use was lower (2.0% (95% CI 0.8–4.4) in 2018), and the respective increase from 2012 levels  was very subtle. Similarly, an increase in e-cigarette use over the study period was observed for non-smoking men. In 2018, 5.2% (95% CI 3.3–7.8) of non-smoking men used e-cigarettes regularly compared to just 1.1% (95% CI 0.4–2.8) in 2012. For non-smoking women, the respective change was from 0.8% (95% CI 0.2–2.3) in 2012 to 2.7% (95% CI 1.3–5.2) in 2018.The sociodemographic and temporal variations in e-cigarette use are given in Among smokers, e-cigarette use was significantly higher among men compared to women  and in the youngest age group compared to 45–64-year-olds . Compared to 2012, the relative odds for e-cigarette use among smokers were highest in 2014 .vs. 45–64-year old) and among those with secondary (vs. tertiary) education, but differences in e-cigarette use across the genders were non-significant. Compared to 2012, the use of e-cigarettes was higher in later study years.Among ex-smokers, higher relative odds for e-cigarette use were found in younger age groups  beforehand to ensure the robustness of the results. Regarding the smoking status, e-cigarette use was very low among respondents who had never smoked. Respective analysis of this subgroup was therefore not performed. The methodological consistency of the survey data could be considered as a strength of the study, but the overall response rates (ranging from 51% in 2018 to 62% in 2012) have been declining across the survey years. We used population weighting to assure data representativeness and reduce the potential non-response bias. Although the latter cannot be fully avoided, a previous study using the same survey data demonstrated that response bias had a minimal effect on prevalence indicators .The results indicate that e-cigarette use has increased significantly since 2012 among men irrespective of smoking status. Although the same period saw a substantial decline in smoking prevalence , the concurrent but opposite trends of smoking and e-cigarette use in Estonia cannot be interpreted causally in our data. However, the decline in smoking prevalence surpassed the increase in e-cigarette use in absolute terms. Despite this, the relative change in e-cigarette use—nearly a three-fold increase among smoking men and almost a five-fold increase in non-smokers—is noteworthy.The prevalence is also slightly higher than in other comparable studies. For example, a study from the United States  found thThe use of e-cigarettes peaked in 2014 and has since then been declining . This is explained by deregulating the sale of e-cigarettes and refill liquids in 2013 . These wIn addition to the previously described period effects, the use of e-cigarettes was patterned by gender , age, and education . Although previous studies have shown mixed findings regarding gender differences in e-cigarette use ,17, we fThe use of e-cigarettes has increased substantially during 2012–2018 among adult men in Estonia. Despite the opposite trend found for smoking prevalence, it is not certain that the increasing popularity of e-cigarettes explains the decrease in smoking rates in Estonia. However, the e-cigarette use varies greatly by smoking status and sociodemographic background. The higher e-cigarette use among young adults calls for a timely and targeted tobacco policy to alleviate the harm of e-cigarette use from the public health perspective.


Nature Communications 10.1038/s41467-020-15005-5, published online 12 March 2020.Correction to: https://zenodo.org/record/3607042#.X8qgac1Ki70 in the PDF and HTML versions of the Article.The original data availability statement for this Article incorrectly listed a link to data file. This has now been corrected to The original version of Table 1, numerical value in the third row from bottom and third column was incorrectly listed The original version of the Article in ‘Results', ‘waveguide loss reduction’ sub-section, third sentence, incorrectly used Ref. [2] in the exponent, this has now been corrected to


FoxP family of transcription factors is necessary for operant self-learning, an evolutionary conserved form of motor learning. The expression pattern, molecular function and mechanisms of action of the Drosophila FoxP orthologue remain to be elucidated. By editing the genomic locus of FoxP with CRISPR/Cas9, we find that the three different FoxP isoforms are expressed in neurons, but not in glia and that not all neurons express all isoforms. Furthermore, we detect FoxP expression in, e.g. the protocerebral bridge, the fan-shaped body and in motor neurons, but not in the mushroom bodies. Finally, we discover that FoxP expression during development, but not adulthood, is required for normal locomotion and landmark fixation in walking flies. While FoxP expression in the protocerebral bridge and motor neurons is involved in locomotion and landmark fixation, the FoxP gene can be excised from dorsal cluster neurons and mushroom-body Kenyon cells without affecting these behaviours.The  In mamm members  are abun members . In partly FoxP1- members uisition .FOXP1 deletions present with mild mental retardation, delayed onset of walking, gross motor impairments and significant language and speech deficits [FOXP2 cause a severe speech and language disorder characterized by deficits in language processing, verbal dyspraxia and impaired grammatical skills, without affecting other traits severely [FoxP genes in vocal learning appears to be evolutionary conserved as knock-outs of the zebra finch orthologue of human FOXP2 during the critical song learning period alters the structure of the crystallized song in the adults [FoxP is involved in such operant learning [Humans with deficits . Mutatioseverely ,5. The fe adults . Such voe adults –17. It wlearning .forkhead (fkh) gene was identified in the fruit fly Drosophila melanogaster [FoxP family members, only one orthologue of the FoxP subfamily is present in flies (dFoxP). The dFoxP gene gives rise to three different transcripts by alternative splicing [FoxP-isoform A (FoxP-iA), FoxP-isoform B (FoxP-iB) and FoxP-isoform IR  (a). The currently available reports as to the expression pattern of the FoxP gene have been contradictory and nothing is known as to whether the different isoforms are differentially expressed in different cell types. To resolve these issues we have tagged the endogenous FoxP gene, analysed the isoform-specific expression patterns and compared them with the expression of a selection of cell-type-specific markers.The original nogaster , where msplicing ,18,20: FoxP-iIR) a. The cuFoxP gene not only show impairments in operant learning, but also in motor coordination and performance of inborn behaviours [FoxP expression at what developmental stage(s) for normal locomotor behaviour. We therefore knocked out the FoxP gene in a spatiotemporally controlled manner and analysed spatial and temporal parameters of locomotor behaviour in the resulting mutants in Buridan's paradigm.Flies with a mutated haviours ,20–22. W2.2.1.ts [FoxP-iB-Gal4 and FoxP-LexA driver line, respectively, were crossed with the appropriate effector lines containing different GFP or RFP variants (UAS-t:gRNA(4xFoxP), this effector line was always first crossed with a UAS-Cas9 line, and the resulting double-effector offspring with the appropriate driver line for each experiment .Fly stocks were maintained at 18°C . Before ts ,24 that variants . For behxxx-Gal4>UAS-Cas9, without gRNAs) and the other drives expression only of the gRNAs  without Cas9). In this fashion, each strain not only controls for potential insertion effects of the transgenes used, but also for potential detrimental effects of expressing the components alone, irrespective of the excision of the target gene.For local knock-out experiments, two genetic constructs need to be brought together for the method to work effectively. The endonuclease Cas9 needs to be present as well as the guide RNA (gRNA) to provide a target for the nuclease. Hence, the appropriate control groups express only one component of the CRISPR/Cas9 combination. One line drives expression only of the Cas9 endonuclease .The transcript and protein sequences of the different FoxP isoforms were downloaded from ate tool a.2.3.FoxP locus [FoxP-iB-Gal4 and FoxP-LexA), one mutant line (FoxP-KO) and one effector line (UAS-t:gRNA(4xFoxP)).We used CRISPR/Cas9 Homology-Directed Repair (HDR) to edit the xP locus  and genexP locus . We crea2.3.1.To create an isoform-specific driver line, we inserted a Gal4 sequence into exon 8, which is specific to isoform B. Two 1 kb homology fragments were PCR-amplified  and digested with the appropriate restriction enzymes  to be ligated in the pT-GEM(0)  vector  which co2.3.2.FoxP isoforms, we inserted a LexA sequence into exon 3. Two 1 kb homology fragments were PCR-amplified  and digested with the appropriate restriction enzymes  to be ligated in the pT-GEM(0) vector which contained a LexA sequence and a 3xP3-RFP-SV40 sequence for selection of transformants. The gRNA sequences used are: sense 5′-CTTCGGGTCGGCCTATGCTATTTA-3′, asense 5′-AAACTAAATAGCATAGGCCGACCC-3′. They were annealed and were cloned inside a pU6-gRNA vector previously digested with BbsI restriction enzyme.To create a driver line that reflects expression of all 2.3.3.FoxP gene, we removed part of exon 1, the complete exon 2 and part of exon 3. Two 1 kb homology fragments were PCR-amplified  and digested with the appropriate restriction enzymes  to be ligated in the pHD-DsRed-attP  vector which contains a 3xP3-DsRed sequence for selection of transformants. The gRNA sequences used are: gRNA1 sense 5′-CTTCGCGGATGATAGTACTTCCGCA-3′, asense 5′-AAACTGCGGAAGTACTATCATCCGC-3′; gRNA2 sense 5′-CTTCGAAGGACGTGCCCGGAAGAGA-3′, asense 5′-AAACTCTCTTCCGGGCACGTCCTTC-3′. They were annealed and were cloned inside a pU6-gRNA vector previously digested with BbsI restriction enzyme.To prevent expression of any isoform of the 2.3.4.To create an isoform-unspecific conditional effector line we phosphorylated and annealed three sets of oligos  that were cloned with a single Gibson Assembly reaction in a pCFD6  vector  which waVas/Cas9; for the FoxP-iB-Gal4, FoxP-LexA and FoxP-KO and Integrase(x);;AttP2 for UAS-t:gRNA(4xFoxP)). The resulting transformants were crossed two times with the balanced flies w-;; D3/TM3, Sb.After the constructs were created, they were injected into early embryos . For larval staining, third instar larvae were selected, dissected in 0.01% PBST and fixed in 4% PFA at room temperature (RT) for 30 min. Clean brains were washed three times in 0.01% PBST for a total time of 45 min and then blocked with 10% normal goat serum (NGS) for 1 h. Subsequently, the brains were incubated with the appropriate primary antibody for one to two nights at 4°C .Three to 6 day-old adults were fixed in 4% paraformaldehyde (PFA) at 4°C for 2 h and dissected in 0.01% phosphate-buffered saline with Tritons at 4°C . After ts at 4°C  for 5–7 2.5.mageJ software  [UAS-Stinger-GFP stacks, using the tool for spots counting. For the FoxP-iB-Gal4/FoxP-LexA count (b), five brains were counted for each genotype at both larval (3rd instar) and adult (2–3 days old) stages. The colocalization analysis was performed with the ImageJ Colocalization Threshold tool (Tony Collins and Daniel James White) (b). The three-dimensional rendering (b) was performed with IMARIS 9.0 software on the Drosophila standard brain from https://www.virtualflybrain.org.All images were acquired with a Leica SP8 confocal microscope (RRID: SCR_018169), images were scanned at a frame size of 1024 × 1024 pixels at 200 or 100 Hz. The objectives were 20× dry and 20×/40×/60× oil immersion. Images were processed with I_003070) , only gexA count b, five bs White) b. The thendering b was per2.6.d and 4e). We extracted RNA from 20 flies for each genotype , following the TriFast™ protocol from peqlab (a VWR company) . The RNA was subsequently transcribed into cDNA using the OneStep RT-PCR Kit from QIAGEN  with the following thermocycler programme: 42°C for 2 min, 4°C pause until manual restart at 42°C for 30 min, 95°C for 3 min and finally 10°C ∞. Subsequently, we performed the qPCR. Primer sequences were identical to those used by Mendoza et al. [2O. As reference, we used the housekeeping gene rp49 , while as a negative control we used the same reaction mix without cDNA. The qPCR reaction programme used was: 95°C for 2 min, 95°C for 10 s, 60°C for 10 s, 65°C for 30 s (from step 2 to 4 × 39 rounds), 95°C for 10 s, and finally from 65°C to 95°C for +0.5°C/5 s. The experiments were repeated two to four times.The knock-out efficiency was assessed using RT-qPCR figures d and 4e.a et al. . For the2.7.a) consists of a round platform with a diameter of 117 mm which is surrounded by a water-filled moat. The platform is situated at the bottom of a uniformly illuminated white cylinder, 313 mm in height and 293 mm in diameter [2 anesthesia. After one night recovery at 25°C, they were tested in Buridan's paradigm for 15 min (doi:10.17504/protocols.io.c7vzn5). The position of the fly is recorded by a camera (Logitech Quickcam Pro 9000) connected to a computer running our BuriTrack software (http://buridan.sourceforge.net.)All behavioural experiments were performed in Buridan's paradigm (RRID: SCR_006331) . In thisfigure 6https://github.com/jcolomb/CeTrAn) extracts a variety of parameters from the scored trajectories. From the parameters extracted by CeTrAn, we used the temporal parameters median speed , distance travelled, number of walks (sections of the trajectory which connect the platform areas closest to the two stripes) and activity time , as well as the spatial parameters stripe deviation  and meander (a measure of the tortuosity of the fly's trajectory). Transition plots visualize the areas on the platform that the flies most frequently visited. More details in [The analysis software CeTrAn  , flies were raised at 18°C, moved to 30°C for 12 h (embryos) or 48 h (pupae and adults) and subsequently left at 25°C for the rest of the development (embryos and pupae) or overnight for recovery (adults) before testing.For the experiment involving 2.8.F-test. In the absence of significantly different variances, we used Student's t-tests (two-tailed) or one way ANOVAs followed by Tukey's post hoc test for multiple comparisons. If the F-test was significant at p < 0.005 (see below), we used a Mann–Whitney U-test or a Kruskal–Wallis ANOVA followed by Dunn's post hoc test for multiple comparisons. Alpha values were set to 0.5%, in order to reduce the chances of false-positives, following the arguments detailed in [All graphs were created and statistical analysis was performed using GraphPad Prism 6  software. Sample variances were compared with an ailed in , where BN ∼ 20. We then used these results to perform a power analysis for the subsequent experiments. We found that effect sizes such as those exhibited in the speed, meander or stripe fixation parameters required a sample size of up to 18 to reach 80% statistical power at an alpha of 0.5% [p < 0.005 was considered significant. Data are expressed as averages ± s.e.m. or averages ± s.d., and each case is indicated in the legend of each figure.The initial behavioural experiments  were car of 0.5% , while e3.3.1.FoxP transcription factor binds DNA with the forkhead (FH) box domain . The gene consists of seven introns and eight exons. The FH box is split into different segments spanning exons 6, 7 and 8. The last two exons (7 and 8) are subjected to alternative splicing, leading to two different protein isoforms: isoform A (FoxP-iA), which results from splicing exon 6 to exon 7, and isoform B (FoxP-iB) where instead exon 8 is spliced to exon 6. A third, intron-retention isoform is transcribed by failing to splice the intron between exon 6 and 7 out (FoxP-iIR). While the first two isoforms contain a complete and putatively functioning FH box , the putative FoxP-iIR FH box appears to be truncated due to a stop codon in the intron sequence , putting the transcription factor function of this isoform in doubt. Of the three FoxP isoforms, FoxP-iB was most directly associated with the learning phenotype discovered by Mendoza et al. [FoxP-iB (figure 1a). This insertion leads to the expression of the Gal4 transcription factor only in FoxP-iB positive cells. At the same time, the insertion also disrupts the FH box DNA-binding domain of the FoxP gene, putatively preventing the FoxP protein to act as a transcription factor, effectively mutating the gene for this function.The a et al. . TherefoFoxP-iB is expressed throughout the whole development of the fly, from embryo (data deposited at doi:10.6084/m9.figshare.12607652) to adult, in both brain and ventral nerve cord (VNC) (b). In 3rd instar larvae, we observe expression in the central brain (but not in the optic lobes) and in the anterior portion of the VNC. In the adult nervous system, the main neuropil expression domains comprise protocerebral bridge, gnathal ganglia , vest, saddle, noduli and superior medial protocerebrum. GFP-positive cell body clusters could be found in the cortex of the central brain and around the optic lobes (b).Observing Gal4 expression with different green fluorescent proteins (GFPs) under control of the UAS promoter , revealed that rd (VNC) b. In 3rdic lobes b.iB-specific driver line to the staining of an available isoform-unspecific polyclonal antibody [c). The cells only stained for the FoxP antibody and not for GFP are presumably cells expressing the other FoxP isoforms  neurons, neither with our driver line, nor with the antibody.We next validated the expression pattern of our antibody . We obseFoxP gene, we measured mRNA levels of all three isoforms with RT-qPCR (d). With one of the primers placed over the Gal4 insertion site, we observed approximately half the wild-type FoxP-iB expression levels in heterozygous animals, while wild-type FoxP-iB expression was essentially abolished in the homozygous transgenes. We did not observe any significant changes in the other two isoforms. However, this disruption of the FoxP-iB isoform did not have an effect on the development of the FoxP-positive neurons, as both heterozygous and homozygous FoxP-iB-Gal4 mutants showed the same number of GFP-labelled cell nuclei (data deposited at doi:10.6084/m9.figshare.12932705).Postulating that our transgene disrupted expression of the  RT-qPCR d. With o3.2.FoxP involved in learning and expression patterns suggesting neuronal expression  and REPO . At both developmental stages, the two stainings reveal exclusive FoxP-iB-mediated GFP colocalization with ELAV without any colocalization with REPO , suggesting that FoxP-iB is expressed exclusively in neurons. These data are consistent with results published previously [With pression , we inveeviously ,36, valiFoxP-iB is expressed. Using a variety of antibodies (10.6084/m9.figshare.12607712) used as markers for different neuronal cell types we detected FoxP-iB expression in most of the cell types investigated. For technical reasons, we stained adult brains only with the anti-TH antibody, while the remaining markers were used on larval nervous systems. Extensive colocalization was observed with p-SMAD1/5 (a motor neuron marker) in the VNC but not in the central brain (CB) (a). Some FoxP-iB neurons were positive for ChAT (cholinergic) or GABA (inhibitory) both in the VNC and in the CB . Finally, a few FoxP-iB positive neurons were found to colocalize with tyrosine hydroxylase (dopaminergic neurons) in the CB only (d). These data are consistent with the study performed by Schatton et al. [doi:10.6084/m9.figshare.12607643), a marker for photoreceptor cells [We next investigated in more detail the types of neurons in which ain (CB) a. Some Fn the CB b,c. Fina CB only d. These n et al.  in honeyor cells .Figure 3.3.FoxP-iB-specific driver line was reporting (b), we created a second driver line, designed to drive expression in all FoxP cells, irrespective of isoform. We inserted a sequence for the bacterial LexA [figure 4a; raw data deposited at doi:10.6084/m9.figshare.12607730). Comparing Stinger-GFP expression from each driver line revealed a more expansive pattern for the isoform-unspecific driver (figure 4b), one that matches very well with the FoxP antibody staining (b). This visual impression was corroborated by a quantification of stained nuclei comparing between both drivers (figure 4c). This quantification allowed us to trace the proliferation of FoxP cells from around 500 in 3rd instar larvae to around 1800 in 3 day-old adults. By contrast, there are only about 300 cells expressing FoxP-iB in the 3rd larval instar and only around 1300 in 3 day-old adults. We noticed that the largest differences in terms of cell number between FoxP-LexA and FoxP-iB-Gal4 flies (both larvae and adults) were found in the CB, while the VNC numbers differed considerably less. Taken together, in 3rd instar larvae and in 3 day-old adults, 66% and 65%, respectively, of the total number of FoxP neurons in the Drosophila nervous system express FoxP-iB.As with our previous insertion, also this one was expected to disrupt expression of the FoxP gene. To investigate the extent of this disruption on the mRNA level, we again performed RT-qPCR. As expected, this insertion affected all isoforms. In heterozygous flies, the expression level was increased, while in homozygous flies it was decreased (figure 4e). It is important to note that here, in contrast to the FoxP-iB insertion, all primers are binding sequences downstream of the insertion site  and so are amplifying all transcripts, with or without the insertion. Possibly, the FoxP protein is involved in its own regulation such that in heterozygotes, the missing gene copy leads to a compensatory increase in transcription rate, but in homozygous mutant animals without any FoxP protein, only basal expression levels remain.As the antibody staining against the FoxP protein indicated more cells expressing FoxP than our eporting b, we creial LexA  transcristaining b. This vion site a,d and sLexAop-RFP;UAS-CD8-GFP) and analysed their patterns in adults (doi:10.6084/m9.figshare.12607763). In this way, we labelled all FoxP-expressing neurons red and neurons that specifically expressed FoxP-iB green. We used the ‘Colocalization Threshold’ tool from ImageJ, which computes false colours to enhance the comparison between the two driver lines and let the differences stand out . The other two isoforms are expressed in additional areas  compared to FoxP-iB expression .In order to directly compare the expression patterns of our two driver lines, we used them to drive reporter genes fluorescing at different wavelengths .We also crossed this 3.4.FoxP gene do not only affect operant self-learning, for instance, different alleles also affect flight performance and other locomotion behaviours to different degrees [FoxP pleiotropy affecting various innate motor behaviours independently from motor learning, we turned to Buridan's paradigm [a).Mutations in the  degrees ,20,21. Bparadigm ,31 as a FoxP expression , our FoxP-iB-Gal4 insertion shows abnormalities in Buridan's paradigm both in temporal and in spatial parameters (doi:10.6084/m9.figshare.12607769). While the homozygous flies walked more slowly, spent more time at rest and fixed the stripes less strongly than wild-type control flies, heterozygous flies only differed from wild-type flies in their stripe fixation (c). With different effect sizes in each parameter, we selected two representative parameters each for the temporal and the spatial domain, respectively, for comparison of all subsequent lines: walking speed, activity time, meander and stripe fixation.Consistent with previous findings of impaired locomotor behaviour in ed flies ,20,21 anpression d, our Fofixation c. With d3955FoxP mutant, we tested the 3955FoxP mutant flies in Buridan's paradigm and found changes in several temporal parameters, similar to those observed in our driver line (d). However, for the spatial parameters (stripe deviation and meander) only stripe fixation appears normal in these flies, the increased meander indicates that the flies have problems walking straight, despite clearly walking towards the stripes. Thus, in addition to the deficits in operant self-learning and flight performance as reported previously [3955FoxP mutant flies are also deficient in several temporal and one spatial parameter of walking behaviour in Buridan's paradigm. This walking phenotype is consistent with previous findings of walking deficits associated with FoxP manipulations [Because our insertion is located in the same exon as the insertion in the ver line d. Howeveeviously , the Foxulations ,21, but ulations .4.FoxP-iB, which is expressed in about 65% of all FoxP-positive neurons .With such dramatic motor alterations when only  neurons , is remo neurons , it is id before . While tFoxP-iB insertion line, we tested both heterozygous and homozygous FoxP-KO deletion mutants in Buridan's paradigm (c–f). The results of this experiment closely resemble the ones from the FoxP-iB-Gal4 insertion line, with homozygous mutants being both significantly less active (c) and fixating the stripes less strongly than the heterozygous mutants and the wild-type controls . For this allele, the heterozygous FoxP-KO mutants show higher values for all temporal parameters compared to the wild-type controls, while there is no difference in stripe deviation. This trend was already observed in FoxP-iB mutants but failed to reach statistical significance. Thus, the FoxP-KO allele exhibits differential dominance, with recessive modes of inheritance in some traits and, e.g. overdominance in others. A direct comparison of the data from the two homozygous alleles (FoxP-iB and FoxP-KO) showed no significant differences (f). Thus, removing the other FoxP isoforms had few effects beyond the consequences of removing only FoxP-iB alone.Analogous to the behavioural characterization in the paradigm c–f. The s active c and fixcontrols d,e. For ferences f. Thus, 4.1.FoxP in the fly's nervous system . The four guide RNAs (gRNA) each target a different section of the FoxP gene . If expressed together with the endonuclease Cas9, this effector efficiently mutates the targeted gene [elav-Gal4 driver and monitoring FoxP expression with the FoxP antibody used before (figure 8b). Flies with this pan-neuronal excision of the FoxP gene (FoxP-cKO) were also tested in Buridan's paradigm and showed even more severe impairments than flies homozygous for a constitutive deletion of the gene (figure 8c). In fact, the mutated flies walked so little, that analysis of spatial parameters was not meaningful (figure 8d). To allow for temporal control of transgene expression, we also validated the use of the temperature-sensitive suppressor of Gal4, Gal80ts (figure 8e). The constitutively expressed Gal80ts prevents Gal4 from activating transcription of the UAS-controlled transgenes until the temperature is shifted from 18°C to 30°C, at which point the repressor becomes inactivated and Gal4-mediated transcription commences [figure 8f), but also on the behavioural level . In both experiments, the effects of the manipulations were so severe, that it was not possible to reach the target sample size of 18.Given the patchy expression pattern of  figures –5 and th figures  and 7, wted gene . We valiommences ,24. Usin4.2.et al. [FoxP-iB expression pattern suggests that at least some of these DCNs express FoxP . Therefore, we used ato-Gal4 to excise the FoxP gene specifically in DCNs. Remarkably, this manipulation did not have any effect on the flies' behaviour in Buridan's paradigm (figure 9b).Recently, Linneweber et al.  describeess FoxP . Compari in DCNs , we obseFoxP expression in the MB Kenyon cells themselves. Finally, there are two reports that expressing anti-FoxP RNAi constructs exclusively in the MBs can have behavioural effects [FoxP mRNA, see below), we deleted the FoxP gene from MB Kenyon cells using the ok107-Gal4 driver and tested the flies in Buridan's paradigm. We did not detect any differences to control flies in these experiments (c).The insect MBs are not only known as a centre for olfactory learning and memory e.g. –50, they effects ,35. For  effects  nor us beriments c.FoxP in motor neurons, besides FoxP expression there  led to a significant alteration in locomotor activity in Buridan's paradigm, both for spatial and for temporal parameters .There are two reasons for knocking out on there : first, on there  and 7. Sre . Removing the FoxP gene exclusively in these neurons led to a significant reduction of locomotor activity (d) as well as a reduction in stripe fixation and to more tortuous trajectories (e). In fact, the stripe deviation increased to an extent that it can no longer be distinguished statistically from a random walk at the 0.5% level .Perhaps the most prevalent l bridge . The dri neurons c. Removiactivity d as wellectories e. In fac4.3.FoxP being a transcription factor active throughout development and particularly important during pupal development [FoxP in all neurons by adding the Gal4 repressor Gal80ts to our pan-neuronal FoxP-cKO  and treating the flies with a 48 h 30°C heat treatment during the early pupal stage. This regime did not affect walking behaviour in Buridan's paradigm (figure 11b). Shifting the temperature treatment to immediately after eclosion also did not affect the flies' behaviour in Buridan's paradigm (figure 11c). Taken together, these data indicate that FoxP is required for the proper development of, for instance, motor neurons and protocerebral bridge neurons, but once these circuitries are in place, FoxP expression does not appear to have any immediate mechanistic role in locomotion anymore.With elopment ,62, we k5.Drosophila FoxP gene in order to better understand the expression patterns of the FoxP isoforms and their involvement in behaviour. We have discovered that the isoforms differ with respect to their expression in neuronal tissue. For instance, we found isoform B (FoxP-iB) expression in neuropil areas such as the superior medial protocerebrum, the protocerebral bridge, the noduli, the vest, the saddle, the gnathal ganglia and the medulla, while areas such as the antennal lobes, the fan-shaped body, the lobula and a glomerulus of the posterior ventrolateral protocerebrum contain other FoxP isoforms but not isoform B (summarized in doi:10.6084/m9.figshare.12607862). We also corroborated previous results [FoxP is expressed in a large variety of neuronal cell types  or in MB Kenyon cells . The Lawton et al. description of the FoxP expression pattern as a small number of neurons distributed in various areas of the brain, particularly in the protocerebral bridge, matches our results and those of [The exact expression pattern of hemistry  . However, since they also used insertion of a transgene, their expression pattern, analogous to that of a promoter fragment Gal4 line, may potentially be subject to local effects where the fosmid with the tagged FoxP was inserted. Castells-Nobau and colleagues [et al. [et al. [b).The fourth and latest study reporting on osophila  avoided a fosmid , intendelleagues  also use [et al.  to valid [et al.  and our Drosophila, we used CRISPR/Cas9 with homology-directed repair to tag FoxP in situ, avoiding both the potential local insertion effects of the previous approaches and without disrupting the complex regulation that may occur from more distant parts in the genome. For instance, in human cells, there are at least 18 different genomic regions that are in physical contact with the FOXP2 promoter, some of which act as enhancers [FoxP locus were inserted in a different genomic region as in [FoxP-KO approaches. This converging evidence from four different methods used in three different laboratories suggests that FoxP is expressed in about 1800 neurons in the fly nervous system, of which about 500 are located in the ventral nerve cord. Expression in the brain is widespread with both localized clusters and individual neurons (b) across a variety of neuronal cell types. Notably, the four methods also agree that there is no detectable FoxP expression in the adult or larval MBs. By contrast, in honey bees, there is converging evidence of FoxP expression in the MBs [In an attempt to eliminate, the last source of error for determining the expression pattern of FoxP in nhancers . The effon as in . Intereson as in  and the on as in  agree bo neurons b across  the MBs .5.2.FoxP expression in the MBs  when there is no FoxP protein detectable there.This comparison of our data with the literature prompts the question why two different promoter fragment approaches ,62 suggeet al. used the classic hsp70-based pGaTB vector [et al. as well as Schatton and Scharff used the more modern Drosophila synthetic core promoter (DSCP)-based pBPGUw vector [A first observation is that Lawton B vector  to creatw vector . The twow vector .FoxP RNA can be detected in more than 4100 brain cells, likely overcounting the actual FoxP expression more than threefold. For instance, FoxP RNA is detected in over 1000 glial cells where none of the published studies has ever detected any FoxP expression , which in wild-type animals rarely leads to any physiologically relevant FoxP protein levels in these cells. Only when gene expression is enhanced by combining some arbitrary promoter fragments with genetically engineered constructs designed to maximize Gal4 yield such as the pBPGUw vector, such transient, low-abundance mRNAs may be amplified to a detectable level.Taking these two observations together, it becomes plausible that there may be transient, low-level FoxP RNA in MB Kenyon cells: the transcript they detected may have been present and occupied by ribosomes, but ribosomal occupancy does not automatically entail translation [et al. failed to detect all those much more strongly expressing and numerous neurons outside of the MBs. All of the above is consistent with other insect species showing FoxP expression on the protein level in their MBs [These considerations may also help explain why the ribosome-based method of  was ablenslation . It remaheir MBs , as onlyThe stochasticity of gene expression is a well-known fact –79 and k5.3.FoxP-iB, is only expressed in about 65% of all FoxP-positive neurons. The remainder express either FoxP-iA or FoxP-iIR or both. Neurons expressing only non-iB isoforms are localized in the antennal lobes, the fan-shaped body, the lobula and a glomerulus of the posterior ventrolateral protocerebrum , CRISPR/Cas9-mediated genome editing lends itself as the method of choice for this task. Practical considerations when designing multi-target gRNAs for FoxP prompted us to begin testing the CRISPR/Cas9 system as an alternative to RNAi with an isoform-unspecific approach first, keeping the isoform-specific approach for a time when we have collected more experience in this technique. In a first proof-of-principle, we used CRISPR/Cas9 to remove FoxP from MB Kenyon cells, DCNs, motor neurons and the protocerebral bridge.Taken together, the results available to-date reveal et al. [FoxP. As detailed above, two groups have reported FoxP expression in the MBs and it appears that some transcript can be found in MB Kenyon cells. With a substantial walking defect both in 3955FoxP mutant flies . There are two possible reasons for this lack of an effect of our manipulation: either FoxP protein is not present in MBs or it is not important in MBs for walking. While at this point we are not able to decide between these two options, our expression data concurring with those from previous studies [FoxP expression in the MBs [3955FoxP mutant flies despite testing for such effects. Motor aberrations as those described here and in other FoxP manipulations [et al. [MBs have been shown to affect both spatial and temporal aspects of locomotion e.g. –60 and Cet al.  reportedpression ) and in on [FoxP , togethenormally c. There  studies ,21 sugge the MBs  did not ulations –22,36 co [et al. .FoxP from DCNs showed no effect (b), despite abundant FoxP expression in DCNs (a). It is possible that a potential effect in stripe fixation may have been masked by already somewhat low fixation in both control strains. On the other hand, even at such control fixation levels, significant increases in stripe deviation can be obtained (e.g. a). Before this is resolved, one explanation is that FoxP is not required in these neurons for landmark fixation in Buridan's paradigm, while the neurons themselves are required.DCNs were recently shown to be involved in the spatial component (landmark fixation) of walking in Buridan's paradigm , but remo effect b, despit in DCNs a. It is ned e.g. a. BeforeFoxP in motor neurons (a), we considered these neurons a prime candidate for a clear FoxP-cKO phenotype. Indeed, removing FoxP specifically from motor neurons only, mimicked the effects of removing the gene constitutively from all cells . It is noteworthy that this manipulation alone was sufficient to affect both temporal and spatial parameters, albeit only one of the two driver lines showed clear-cut results (D42). One would not necessarily expect motor neurons to affect purportedly ‘higher-order’ functions such as landmark fixation. It is possible that the higher tortuosity in the trajectories of the flies where D42 was used to drive our UAS-gRNA construct is largely responsible for the greater angular deviation from the landmarks in these flies and that this tortuosity, in turn is caused by the missing FoxP in motor neurons. Alternatively, D42 is also driving in non-motor neurons where FoxP is responsible for landmark fixation. The driver line C380 showed similar trends, albeit not quite statistically significant at our alpha value of 0.5%, suggesting that potentially the increased meander parameter may be caused by motor neurons lacking FoxP.Motor neurons are involved in all aspects of behaviour and have been shown to be important for operant self-learning . With ab neurons a, we conll cells a,b. It iFoxP-positive neuropil, it has also been reported to be involved in temporal aspects of walking [FoxP from a small group of brain neurons innervating the protocerebral bridge, phenocopies constitutive FoxP mutants.The protocerebral bridge is not only the arguably most conspicuous  walking –95. More walking ,96–98. STaken together, the motor neuron and protocerebral bridge results suggest that both sets of neurons serve their locomotor function in sequence. At this point, it is unclear which set of neurons precedes the other in this sequence.5.4.3.FoxP family of transcription factors acts during development in a variety of tissues [FoxP expression serves any specific function. A recent study in transgenic mice in operant conditioning and motor learning tasks showed postnatal knock-out of FOXP2 in cerebellar and striatal neurons affected leverpressing and cerebellar knock-out also affected motor-learning [FoxP family member does serve a postnatal function that is independent of brain development . Also in birds, evidence has been accumulating that adult FoxP expression serves a song plasticity function [FoxP expression in the adult, as long as FoxP expression remains unaltered during larval development. Future research on the role of FoxP in locomotion and landmark fixation hence needs to focus on the larval development before pupation.There is ample evidence that the  tissues ,99,100. learning . At leasfunction –106. Our